Explanation The ectodomain of the plasma membrane ecto-enzyme CD38 functions as both an NAD glycohydrolase and an ADP-ribosyl cyclase by catalyzing, respectively, the conversion of NAD to nicotinamide and ADP-ribose or cyclic ADP-ribose. CD38 is attracting particular attention in cancer therapy. An anti-CD38 monoclonal antibody (daratumumab) was approved for treatment of patients with multiple myeloma. However, the role of CD38 in non-hematological malignancies has not been explored. Previously, we reported that ADP-ribose-acceptor hydrolase (ARH)-1 deficiency in mice was associated with tumor development. In the present study, we found that in wild-type and ARH1-deficient mice deletion of the CD38 gene reduced tumor formation. 1. Effects of CD38 on tumor formation in ARH1-deficient mice As described previously, ARH1 deficiency contributes to spontaneous tumor formation in multiple organs. ARH1/CD38 double-knockout mice were generated to evaluate the effect of CD38 on tumorigenesis. In each instance, ARH1 and CD38 genotype and protein expression were confirmed. CD38-/-, ARH1-/- and ARH1-/-/CD38-/- mice were viable and fertile. Tumor development in WT, ARH1-/-, CD38-/- and ARH1-/-/CD38-/- mice was monitored for as long as 18 months. As expected, based on prior studies, ARH1-/- mice developed a variety of malignancies (lymphoma, adenocarcinoma and hemangio/histolytic sarcoma). Tumors were seen in both male and female WT (0.99% vs 6.93%), ARH1-/-(3.57% vs 18.75%), CD38-/- (0.81% vs 0) and ARH1-/-/CD38-/- (1.23% vs 1.23%) mice. The total tumor occurrence was significantly higher in ARH1-/- mice than in their wild-type (WT) counterparts (22.32% vs 7.92%, p<0.05). CD38-/- (0.81%) and ARH1-/-/CD38-/- (2.46%) showed significant reductions in spontaneous tumor formation, compared to respectively ARH1-/- or WT mice. Our results suggested that CD38 deletion suppressed tumor development in WT or ARH1-/- mice. 2. CD38 deficiency upregulated NAD+ level in mice CD38 is believed to be the major NAD glycohydrolase (NADase) in mammalian cells, as indicated by the markedly decreased NADase activity and increased -NAD levels in CD38-deficient mice. We investigated the association between NAD level and CD38 deficiency. Compared to wild-type mice, CD38-/- mice as well as ARH1-/-/CD38-/- mice have, in multiple organs, significantly higher levels of NAD+ including brain (342.753.1%, 410.050.49% vs 10018.1%), heart (232.413.6%, 174.510.85 vs 10020.7%) and lung (322.789.6%, 292.439.1% vs 10025.6%). No significant differences were found in NAD+ levels between wild-type and ARH1-/- mice (brain 75.243.1%, heart 68.710.1%, lung 98.935.8%). 3. CD38 expression by human lung carcinoma cell lines and specimens We performed comprehensive examination of CD38 expression from transcript to protein level in both human lung cancer cell lines and paired human lung cancers and normal lung samples. A panel of lung cancer cell lines was examined for CD38 mRNA expression. We observed most lung cancer cell lines had higher copy numbers of CD38 mRNA than normal human respiratory epithelial cell lines. We next searched for CD38 and ARH1 gene expression using the Oncomine database. Most studies showed that CD38 mRNA levels were significantly upregulated in human lung carcinoma compared to normal lung tissues, while ARH1 mRNA was down-regulated significantly in patients with lung cancer. Enhanced expression of CD38 protein in several lung cancer cell lines was confirmed by immunoblotting. In addition, CD38 protein expression was examined in human lung carcinoma samples. Samples from 27 lung cancer patients, including 12 males and 15 females, were collected for analysis of CD38 expression. Compared to adjacent normal tissues, all the tumor tissues were infiltrated with strong CD38-positive cells such as macrophages, plasma cells and lymphocytes. CD38 overexpression in tumors cells were detected in 11 of 27 samples, including adenocarcinoma and squamous cell carcinoma. These data together indicated that in general, CD38 was overexpressed in human lung carcinomas. 4. Knockout of CD38 inhibits lung cancer growth in vitro and in vivo To explore the possible role of CD38 in human tumorigenesis, we generated CD38KO A549 cell lines using CD38 CRISPR/Cas9 KO plasmids. Protein expression was completely ablated in CD38KO A549 cells. Two control, 2 knockout and parental cell lines were used for further experiments. Compared to A549 parental cells, cell proliferation did not differ in CD38KO cell lines, while CD38 deletion led to reduced anchorage-independent colony formation in soft agar and suppressed serum-induced cell invasion in transwell invasion assays. To further evaluate the role of CD38 in tumor progression, we injected the above cells subcutaneously into nude mice. Tumor xenograft growth of CD38-deficent cells (KO1 and KO2) were remarkably slower than those of control cell lines (Con1 and Con2) in both female and male nude mice. Based on the above results, we conclude that CD38 knockout may suppress tumor progression. Summary: CD38 overexpression was found in human lung cancer cell lines and tissues. CD38 deletion inhibited oncogenesis in vitro and in vivo. It is expected that anti-CD38 treatment may have therapeutic potential in lung cancer.